Diagnostic test for schizophrenia

ABSTRACT

A test method is provided for determining whether a person is a schizophrenic, which comprises suitably treating a plurality of test animals, obtaining a sample of the person&#39;&#39;s blood, premixing the blood, or preferably the plasma therefrom with 3,4dimethoxyphenylethylamine, internally administering the mixture to the test animals, and maintaining the animals under aggregated or crowded conditions while observing their behavior for amphetamine-like reaction and lethality as an indication of the presence of schizophrenia in the blood sample donor.

United States Proctor atent [4 1 May 23, 1972 [54] DIAGNOSTIC TEST FORSCHIZOPHRENIA [21] Appl.No.: 744,628

Proctor et a]. (l), The Pharmacologist, Vol. 9, No. 2 p. 238 1967)Proctor et a1. (2), Arch. Int. Pharmacodyn, (1968) Vol. 172, No. 1 pp.95-105 Proctor et a1. (3), Arch. Int. Pharmacodyn, (1968) Vol. 172 No. 1pp. 106-121 Primary Examiner-Albert T. Meyers Assistant Examiner-NormanA. Drezin Attorney-Samuel Kurlandsky ABSTRACT A test method is providedfor determining whether a person is a schizophrenic, which comprisessuitably treating a plurality of test animals, obtaining a sample of thepersons blood, premixing the blood, or preferably the plasma therefromwith 3,4- dimethoxyphenylethylamine, internally administering themixture to the test animals, and maintaining the animals underaggregated or crowded conditions while observing their behavior foramphetamine-like reaction and lethality as an indication of the presenceof schizophrenia in the blood sample donor.

5 Claims, No Drawings DIAGNOSTIC TEST FOR SCI-IIZOPI-IRENIA BACKGROUNDOF THE INVENTION Prior art methods for diagnosing schizophrenia aregenerally psychological in nature, and therefore highly subjective. Itwould consequently be highly desirable to have a positive chemical orclinical test for schizophrenia which would be more objective, moreprecise, and more highly reproducible.

It has been previously demonstrated that there is a urinary excretion of3,4-dimethoxyphenylethylamine (DMPEA) by schizophrenic patients which isnot present in analyses run analogously on urine from non-schizophrenicsubjects. Additionally, the presence of abnormal protein factors havebeen shown to be present in theblood plasma of schizophrenic patients.However, the analyses for the presence of these components per se arenot sufficiently developed to provide a diagnostic test forschizophrenia.

SUMMARY OF THE INVENTION It is an object of the present invention toprovide a diagnostic clinical test for the existence of schizophrenia ina person. It is a further object to provide a test of the type describedwhich is sensitive, precise, accurate, and reproducible. It is a furtherobject to provide a diagnostic test which can be carried out in arelatively short time and which is relatively inexpensive. Additionalobjects and advantages of the invention will be apparent to one skilledin the art, and still other advantages will become apparent hereinafter.

DESCRIPTION OF THE PREFERRED EMBODIMENTS According to the invention, aplurality of test animals are first pretreated with a monoamine oxidaseinhibitor (MAO), as for example, phenylisobutylhydrazine. A sample ofblood is taken from the patient and mixed with DMPEA, and afterincubation, the mixture is injected into a plurality of the test mice,and the mice maintained in the aggregated or crowded state where theyare observed for a period of about 4 hours for -exhibition ofamphetamine-like reaction and lethality. It has been found that when theblood from a schizophrenic person is utilized either as serum orpreferably as plasma, a high degree of positive amphetamine-likereaction and a high degree of lethality of the test animals will beobserved.

Reports containing detailed information with regard to the method of thepresent invention are contained in a report entitled SchizophrenicPlasma induction of an Action of DMPEA by Charles D. Proctor et al. inThe Pharmacologist (Vol. 9, No. 2, p. 238,) Fall Issue (1967), and alsotwo reports in Archives Internationale: de Pharmacodynamie et deTherapie, Vol. 172, No. 1, March, 1968, Brussels Belgium, one entitledAN IN- FLUENCE OF BLOOD PLASMA FROM SCHIZOPHRENICS ON AN ACTION OF3,4-DIMETHOX- YPHENYLETHYLAMINE," pp. 95-105, and the other entitledFACTORS AFFECTING AN INFLUENCE OF BLOOD PLASMA FROM SCHIZOPHRENICS ON ANACTION OF 3,4-DlMETl-1OXYPHENYLETl-IYLAMINE, pp. 106-121, the entirecontents of which reports are hereby incorporated herein by reference.

The following examples are given by way of illustration only and are notto be construed as limiting.

Example 1: Test procedure.

Five to ten Webster mice (20-30 gm) are injected intraperitoneally with30 mg/kg JB835 (phenylisobutylhydrazine) in either the hydrochloride orsulfate, or any other suitable form 3 hours prior to further testinjections. Intraperitoneal injection here and elsewhere in the test isat volume level of 0.01 ml/gm. wt. of mouse, e.g., JB-835 concentrationin the injection solution is 3 mg/cc in distilled water.

Two and one-half hours later 0.2 ml. of the plasma from the patientbeing tested is added to 1.8 ml. of a solution of 5.56 mg/mlDMPEA(3,4-dimethoxyphenylethylamine in free base form) in distilledwater in a small test tube. Solution volume measurements are made with a1 ml. tuberculin syringe. The

tube is tightly capped at once. The plasma DMPEA solution is placed inan incubator set at 37 C immediately and incubated immediately at 37 Cfor 30 minutes. 0.01 Ml/gram (mouse wt.) of the incubated DMPEA-plasmasolution is injected intraperitoneally into the 13-835 pretreated mice.The mice are aggregated at once by placing them five per compartment inthe compartment(s) of a No. 101 Keystone plastic mouse cage divided bymeans of a four-compartment, Keystone stainless steel cage divider. Cagetop (Keystone model no. 101- std.) must be on the cage and should beweighted down with a ringstand bottom. One-half inch depth of sawdust ispresent in bottom of the compartmentalized cage. In vitro part of testmust be carried out at room temperature of 7074 F. The mice are observedfor 4 hours, observing for amphetaminelike reaction and lethality.Evaluation of quality of the amphetamine-like reaction in the test isperformed as described in Chance, M.R.A., J. Pharmacol. Exptal. Therap.87:214(l946).

Guidelines used in establishing the quality rating for theamphetamine-like reactions in the test are as follows: Very slightpositive reaction: salivation with increase in locomotor activity butwith none of the mice assuming defensive" postures (mainly rearing up ontheir hind legs as described by Chance, ref. vide supra);

Slight positive reaction: characterized by salivation with definiteincrease in locomotor activity but no assumption of defensive postures;

Moderate positive reaction: animals show salivation, increased locomotoractivity, and assumption of defensive postures in some but not all ofthe animals;

Positive reaction: animals show salivation, marked increase in locomotoractivity and development of defensive posture in all of the animals.

Example 2: Clinical tests.

Using the method of Example 1, plasma was taken from the blood of 12acute, typical schizophrenics, six acute rapidly remittingschizophrenics, and 10 non-schizophrenics. Additionally, six trial testswere made substituting 0.9% aqueous NaCl solution for plasma. Rightafter plasma-DMPEA injection the mice were aggregated (Arch. intoPharmacodyn., 163: 79, 1966) and observed for amphetamine-like reaction(ALR) and 4 hour lethality (L). Results showed: with 12 acute, typicalschizophrenics, marked ALR, 100% L; with six acute rapidly remittingschizophrenics, moderate ALR, 20 40% L; with 10 non-schizophrenics, ALR,0% L; six trials substituting 0.9% NaCl for plasma, ALR, 0% L.

Table I below illustrates the effect of removing any one of the threeimportant components necessary to given a positive test according to theinvention.

TABLE I Influence of Deletion of Components on the Effect of the 30mg/kg .IB-835 50 mg/kg DMPEA Schizophrenic Plasma System in AggregatedMice.

1) 18-835 was given intraperitoneally (i.p.) 3 hours prior to DMPEAinjection. DMPEA was given i.p. immediately prior to aggregation of themice. When either JB-835 or DMPEA was omitted. physiological saline wasadministered as a substitute, "sham" injection at analogous volume andtime schedule.

2) Plasma used in both Examples 6 and 7 was from the same blood samplewithdrawn from an unmedicated. acute typical schizophrenic patient.Plasma was added to 5.56 mgJml. DMPEA at 1:9 volume, incubated at 37 C.for 30 minutes right before i.p. injection followed by immediateaggregation of animals. Physiological saline was substituted for plasmawhere indicated.

Statistical Analysis: p value, X3: Example 7 vs. any other group: p0.0005.

The findings which have been presented in Table I appear to support theessentiality of having the intact combination of MAO inhibitor, DMPEAand plasma from a schizophrenic patient in the biological test systemunder study in order to produce an amphetamine-like. reaction andlethality. Consideration of the results reveals that neither of theseresponses were yielded under the following conditions of deletion fromthe three component system in question: absence of the schizophrenicplasma (Example 3); 30 mg/kg .IB-835 pretreatment of animals present butthe other two components absent (Example 4); 50 mg/kg DMPEA given along(Example 5 50 mg/kg DMPEA incubated with schizophrenic plasma and givento mice not pretreated with .1 B835 (Example 6). The lack of response inall of these situations is in marked contrast to the definite responsesobtained with the intact three component system of JB835premedication-incubated DMPEA-schizophrenic plasma injection (Example7). In another experiment (carried out using a schizophrenic plasmadifferent from the one used to supply data in Table I) it was revealedthat incubating the plasma with physiological saline (in lieu of DMPEAsolution), and injecting this into 30 mg/kg JB835 pretreated, aggregatedmice did not produce the responses. This was in spite of the fact thatrunning the test on this plasma with its use making the three componentsintact in the system produced positive amphetamine-like reaction and 80percent lethality (X p 0.002).

Table II below illustrates the effects of aggregation or crowding of thetest animals as opposed to solitary conditions.

TAB LE II Effect of Uncrowding: 30 mg/kg 18-835 50 mg/kg DMPEA TypicalAcute Schizophrenic Blood Plasma System in Aggregated and UnaggregatedMice.

Degree of Amphetaminep value Example Aggregation Like Reaction Leth- X}ality 1"") 8 Crowded 100% p 0.0005 9 Uncrowded, 0%

Solitary 1).15-835 given i.p. 3 hours prior to DMPEA injection.

2) Plasma used in both tests was from the same blood sample withdrawnfrom an unmedicated typical acute schizophrenic patient. Plasma wasadded to 5.56 mg/ml. DMPEA at 1:9 volume, and incubated at 37 C. for 30minutes immediately before i.p. injection followed by immediateaggregation ofanimals.

The DMPEA must be used in the free base form in order that the presenttest be efiective. Weak acid salts, ie.,' acetates may also be used.

Table 111 below illustrates the effect of floor space within which themice are aggregated with respect to test results.

TABLE I11 Effect of Variation in Available Floor Space/Mouse on the18-835 Schizophrenic Plasma*-DMPEA Test Run on Mice Aggregated in Groupsof Five Mice Per Compartment.

Floor Space/ Amphetamine- Lethality Like Example Mouse, cm.2 Reaction (n10) 10 25 cm. 100% l l 50 cm. 12 cm. 50% 13 400 em. slight 10% The sameschizophrenic plasma sample was used in all of the concurrent runs ofthe test shown.

*Example 10 or 1 1. each compared with Example 13:X,.*, p 0.05, eachinstance.

Table IV below shows the results of experiments designed to determinethe effect of time of injection of the MAO inhibitor.

TABLE IV Effect of Time of Injection of 30 mg/kg JB835 Prior toIncubated DMPEA Schizophrenic Plasma Solution* on Test Response toJB-835-DMPEA-Plasma Test in Aggregated Mice.

The same schizophrenic plasma was used in all tests shown. "EitherExample 14 or Example 15 compared separately with each of any of theother Exam les, X p 0.05.

Table V below illustrates the effect of varying the incubation period at37 C in vitro of plasma solution and DMPEA.

TABLE V Effect of lncubation Time at 37 C in Vitro for DMPEA-Schizophrenic Plasma Solution on JB-835-DMPEA-P1asma* Test Response inAggregated Mice.

Time, Min., of Incubation of DMPEA-Plasma Amphetamine- 4 hour SolutionBefore Like Response Lethality Example injection or (n 10)" 20 none 0%21 10 min. very slight 10% 22 15 min. moderate 20% 23 30 min. 100% 24 35min. 100% The same schizophrenic plasma was used in all tests shown."Either Example 23 or 24 compared separately with each of any of theother Examples. X3. p 0.0$.

Table VI below shows results of varying the temperature of 30 minuteincubation of plasma and DMPEA.

(boiling water bath) Table Vll below indicates the effect of varying thedosage of DMPEA.

TABLE VI] Dose of DMPEA yielded from Cone. of DMPEA (free base) ininjection of Solution added Lethality DMPEA-Plasma to Plasma at ExampleSolution Vol. Ratio 9:1 (11 32 mg./kg. 2.22 mg./ml. 0% 33 mgjkg. 2.78mg./ml. 10% 34 mgJkg. 3.34 mg/kg. 10% 35 mg./kg. 4.44 mgJml. 40% 36 50mg./kg. 5.56 mg./ml. l00% 37 60 mgJkg. 6.67 mgJml. 100% 38 70 rug/kg.7.78 mg./ml. i00% Amphetamine-like response occurred over the wholerange of 20 mg/kg 70 mg/kg DMPEA. Significant lethality useful fortesting occurred over the range 40 mg/kg. 70 mg/kg. DMPEA.

According to the findings discussed above, three components are requiredfor elicitation of the responses which have been observed. Theserequirements are: that the aggregated mice used be pretreated with anMAO (monoamine oxidase) inhibitor, as for example, JB-835; that DMPEA bepresent in the system; and that suitable means be afforded for the DMPEAto interact with some factor or factors present in the blood ofschizophrenic patients but not present in blood taken fromnon-schizophrenics. Aside from the inference of CNS (central nervoussystem) action derived from the requisite inclusion of the MAO inhibitorin the system, the requisite nature of all of these three components toelicitation of response in the biological system presents otherimplications. The critical requirement for interaction between DMPEA andsome schizophrenic plasma factor (or factors), considered together withthe probable CNS nature of the test response, is not incompatible withthe hypothesis that the interaction arranges for increased transport ofDMPEA across the blood-brain barrier to effect response. An alternativepossibility, likewise neither proved nor disproved by the present data,is that some agent or system present in schizophrenic plasma (but not innon-schizophrenic plasma) protects DMPEA from alteration to an inactiveproduct either in the process of incubation or in vivo or both. Thenature of the results establishing essentiality of the three criticalcomponents in the system in order to evoke response certainly tends tosupport rather than detract from the notion that at least a part of theprocess of schizophrenic reaction may be associated with an alteredmetabolism involving DMPEA and production of blood proteins which arespecific (either by presence or by relative level) to the process ofschizophrenic reaction.

Present results indicate that in consideration of the relative potentialdiagnostic utility of the test system for schizophrenic reaction, plasmafrom the subject wil probably have greater utility than will serum. Itwas ascertained in comparative runs of the test, utilizing serum andplasma from the same patient, that qualitatively greater biologicalresponses were obtained with plasma. However, either plasma or serum maybe operatively used.

The present results also indicate that changing the social situation ofmice subjected to this test system to uncrowded solitary confinementcompletely reversed the lethality which was obtained when similarlytreated mice were aggregated.

It is to be understood that the invention is not to be limited to theexact details of operation or exact compounds shown and described, asobvious modifications and equivalents will be apparent to one skilled inthe art, and the invention is therefore to be limited only by the scopeof the appended claims.

I claim:

1. A method for diagnosing schizophrenia in a person comprising thefollowing steps:

I. pretreating a plurality of test animals selected from the groupconsisting of mice, rats, rabbits and guinea pigs by internallyinjecting an amount of a monoamine oxidase inhibitor effective toinhibit monoamine oxidase in said test animals,

2. mixing a blood serum or plasma sample taken from the person with 40mg/kg to 70mg/kg of 3,4-dimethoxyphenylethylamine based on the weight ofsaid test animals and incubating the mixture so prepared for a period ofabout 30 minutes at a temperature of 25to 45 C,

3. internally injecting the mixture (2) into said test animals at least3 hours after the injection of said MAO inhibitor, and

4. observing the animals for a suitable period of time while saidanimals are maintained in aggregated condition to determine whether theydevelop amphetamine-like reactions and lethality as indications of theexistence of schizophrenia in the person from whom said blood sample wastaken.

2. A method according to claim 1, wherein said monoamine oxidaseinhibitor is phenylisobutylhydrazine.

3. A method according to claim I, wherein said animals are mice.

4. A method according to claim 1, wherein said sample is plasma.

5. A method according to claim 1, wherein said sample is serum.

2. A method according to claim 1, wherein said monoamine oxidaseinhibitor is phenylisobutylhydrazine.
 2. mixing a blood serum or plasmasample taken from the person with 40 mg/kg to 70mg/kg of3,4-dimethoxyphenylethylamine based on the weight of said test animalsand incubating the mixture so prepared for a period of about 30 minutesat a temperature of 25*to 45* C,
 3. internally injecting the mixture (2)into said test animals at least 3 hours after the injection of said MAOinhibitor, and
 3. A method according to claim 1, wherein said animalsare mice.
 4. A method according to claim 1, wherein said sample isplasma.
 4. observing the animals for a suitable period of time whilesaid animals are maintained in aggregated condition to determine whetherthey develop amphetamine-like reactions and lethality as indications ofthe existence of schizophrenia in the person from whom said blood samplewas taken.
 5. A method according to claim 1, wherein said sample isserum.